Figure 95: Elution 4 D of RpFabG using cation exchange
Figure 94: Elution 4 C of RpFabG using cation exchange
Figure 93: Elution 4 B of RpFabG using cation exchange
Figure 92: Elution 4 A of RpFabG using cation exchange
Elution 3: 25% Buffer A: 75% Buffer B
Figure 91: Elution 3 D of RpFabG using cation exchange
Figure 90: Elution 3 C of RpFabG using cation exchange
Figure 89: Elution 3 B of RpFabG using cation exchange
Figure 88: Elution 3 A of RpFabG using cation exchange
Elution 2: 50% Buffer A: 50% Buffer B
Figure 87: Elution 2 D of RpFabG using cation exchange
Figure 86: Elution 2 C of RpFabG using cation exchange
Figure 85: Elution 2 B of RpFabG using cation exchange
Figure 84: Elution 2 A of RpFabG using cation exchange
Elution 1: 75% Buffer A: 25% Buffer B
Figure 83: Elution 1 D of RpFabG using cation exnchange
Figure 82: Elution 1 C of RpFabG using cation exnchange
Figure 81: Elution 1 B of RpFabG using cation exnchange
Figure 80: Elution 1A of RpFabG using cation exnchange
The goal concentration was to get as close to .1 mg/ml as possible. The -0.00 concentration indicates that all the contaminants were positively charged, and stuck to the negatively charged resin. This is a highly unlikely occurrence so the rest of the procedure was completed as a safety precaution.
Figure 79: Stop Flow step concentration. All 4 samples had the same concentration
Week 13 & 14 (Nov 18 - Dec 1)
Enzyme Assay The enzyme Assay was unsuccessful. This could be due to contaminants in the sample. Cation exchange will be used to further purify the sample since Nickel column purification was not effective.
Figure 78: Enzyme assay with RpFabG, Custum HEPES Buffer, Water, NADPH, and AAC
Protein Expression
Small cultures went in at 10:30AM, after 8 hours (6:30PM) no growth was apparent. The cultures were left in the shaking incubator over night to see if they needed more time to grow, or if the bacterial colonies were dead. At 7:07, small culture tubes were made for the second plate of bacterial colonies with the RpFabG insert to determine if the plate was still viable. All samples grew up overnight, expression protocol was continued. Two 500mL flasks of LB were used. Flask A failed to grow while Flask B was successful. Sample B was sonicated then sun down, sterile filtered, and purified with the Nickel column. A protein gel was run to determine the purity. Elution 1 had a yield of 17.31mg in 2mL of buffer and Elution 2 had a yield of 11.2mg in 4mL of buffer. Elutions were concentrated into 1 mL and then .5mL was stored with glycerol and .5mL was snap frozen.
Figure 77: RpFabG Elution 2 using Custom HEPES Buffer
Figure 76: RpFabG Elution 1 using Custom HEPES Buffer
Week 11 & 12 (Nov 4 - Nov 17)
Great work Priya, nice assays. -UM Enzyme Assays
Conditions in each of the enzyme assays (1-6) had different conditions. Enzyme assay 1 (Green) began at the same conditions as the assays conducted in figure 64, but was unsuccessful. Since the assay was unsuccessful, calculations were adjusted to account for more enzyme. In assay 2 (Blue) 30 uL of enzyme was used at the start, enzyme was added multiple times in 5 uL increments. Enzyme assay 3 (purple) was adjusted to being with 50 uL of enzyme. Assay 4 (orange) Started with 70 uL of enzyme and more was added multiple times in 10 uL increments. Assay 5 (pink) started with 100uL of enzyme and additional enzyme (50 uL) was added once. Enzyme assay 6 (red) had enzyme added in 50 uL increments for a total of 500 uL. Assay 1-4 were with sample stored in the -80C freezer while assay 5r and 6 were using sample stored with glycerol. All enzyme assays were unsuccessful. This could be attributed to having a contaminated sample or samples with low concentrations. There is a possibility that the enzyme can not be stored for long periods of time and still be functional.
Figure 75: Enzyme Assay on RpFabG. Assays 5 and 6
Figure 74: Enzyme Assay on RpFabG Assays 3 and 4
Figure 73: Enzyme Assay on RpFabG Assay 1 and 2
Enzyme Assays Enzyme assay 1 (Orange) began at the same conditions as the assays conducted in figure 64, but enzyme was added multiple times in 5uL increments. In assay 2 (Pink) 20 uL of enzyme was used at the start. Enzyme assay 3 (green) had the same starting conditions as assay 2 but enzyme (5uL) was added one additional time. Assay 4 (blue) Tris buffer was used instead of HEPES since the assays were not successful. This assay showed that NADPH was being oxidized by the enzyme (RpFabG) since absorbance was decreasing.
Figure 72: Enzyme Assay on RpFabG to validate that results could be recreated on a separate day. Assays 3 and 4
Figure 71: Enzyme Assay on RpFabG to validate that results could be recreated on a separate day. Assays 1 and 2
Virtual Screening
The top 5 ligands from the CB_306 library with a logP below 3 were ordered.
Figure 70: Top 4 Cb 306 compounds with a logp < 3 that were ordered
Week 9 & 10 (Oct 21 - 3- Nov)
Great work! It would be good to include the results table from your ligand virtual screening run, too! -Suman 11/5/13 Virtual Screening
Figure 69. Top 5 NIH Clinical screened compounds
Figure 68. Top screened ligands from In House compound library
Figure 67. Top ligands from HF9 library
Figure 66. Top screened compounds from cb 306
Enzyme Assay
The orange line is the end of the third assay. The green line is NADPH, buffer and water, the blue is Buffer, water, NADPH, and enzyme. The red line is enzyme,ACC, NADPH, water, and buffer. These results show that NADPH is the smaller hill at about 340nm. The next steps will be to attempt to replicate this data and then to vary the amount of NADPH while keeping the amount of AAC constant. Then vary the amount of AAC while keeping the amount of NADPH constant in several enzyme assays.
The absorbance of NADPH decreased as the reaction progressed. NADPH was oxidized to NADP. The graph indicated the assay worked.
Figure 64: Enzyme Assay using sample stored in -80C freezer.
Dilutions for NADPH and AAC were calculated and prepared. Enzyme assay calculations were completed and three assays were conducted. Virtual Screening
Five negative controls and 10 positive controls were obtained using PDB. The SDF files were concatenated into a control library. The library was then lig-prepped using ICM since Maestro was still down and docked using GOLD. The control ligand docking was analyzed and confirmed as accurate. Script files were made to run screening jobs on the Hit Finder library, NIH Clinical, Cb 306, and In House libraries.
Figure 63: RpFabG shown as surface. NADPH shown as sticks. Pose 25 of 30 was the best docked when compared to other FabG structures
The pdb structure of RpFabG was obtained (3F9I). No ligands were docked, so NADPH was extracted from FabG from another organism. An attempt to ligand prep NADPH was made but ICM and Maestro were down. After numerous attempts at different times on different days, NADPH was prepared using ICM since Maestro was still down. NADPH was then docked multiple times using ICM with each attempt resulting in various errors or a low docking score. GOLD was then used to dock NADPH into the active site, generating 30 poses.
Figure 62. NADPH docked in the active site of RpFabG. NADPH shown as sticks, polar contacts shown with black dashes, and RpFabG protein shown as surface
Week 7 & 8 (Oct 7 - Oct 20)
Good captions and brief analysis of your data. Where are you with virtual work? Thank you. -Max 10/21/13 RpFabG Sonication, Spin Down, Purification, Characterization, Concentration, and Storage**
Figure 61: Protein Characterization Gel, Well 1: SKip, Well2: Protein Ladder, Well 3: Elution 1 9/27, Well 4: Elutions 2 9/27, Well 5: Elutions 1A, Well 6: ELution 2A, Well 7: Elution 2A (Again since wrong amount of sample was added to previous well) Well 8: Skip (two samples were added to well), Well 9: ELution 1B, Well 10: Elution 2B
Figure 61: Concentration of 2mL concentrated RpFabG Sample
The two samples saved from the previous week were sonicated, spun down, filtered, and then purified. The concentrations of these elutions are relatively high when compared to past elution samples for RpFabG. This can be attributed to using the custom made HEPES buffer with a high concentration of imidazole instead of the elution buffer. A characterization protein gel was run containing elution 1 and 2 samples. The purified samples were stored for 2 days.The samples were then concentrated to about 2mL, split into two 1mL samples that were stored by snap freezing with liquid nitrogen and glycerol respectively. There is still contamination in sample, but FPLC was skipped this time since all previous attempts failed.
Figure 60: RpFab G Elution 2B after purifying sample using nickel column
Figure 59: RpFab G Elution 1B after purifying sample using nickel column
Figure 58: RpFab G Elution 2A after purifying sample using nickel column
Figure 57: RpFab G Elution 1A after purifying sample using nickel column
Week 5 & 6 (Sept 23 - Oct 6)
Good Captions but try to add an analysis of your data. Thank you. -Max 10/07/2013 10/1/13 Virtual Screening Positive and Negative controls were found for target. Since NADPH was not docked in the original crystal structure, similar structures of FabG were used to dock NADPH and define the active site after completing the ligand prep protocol.
RpFabG Expression RpFabG was expressed using the new expression protocol, Option A. The sample was stored to be sonicated.
9/27-28/13
Protein Expression**
RpFabG sample that was stored after sonication was spun down, purified using the nickel column, and then purified through FPLC. FPLC result show a minimal amount of protein, but not enough to be purified. RpFabG will be expressed again and samples will be purified in the room temperature FPLC machine using a nickel column.
Figure 56: RpFab G Elution 1 FPLC results
Figure 55: RpFab G Elution 1 after purifying sample using nickel column
Figure 54: RpFab G Elution 1 after purifying sample using nickel column
Week 3 & 4 (Sept 9 - Sept 22)
9/16/13 Priya - Good work on purifications. what does your gel look like for these? There doesn't seem to be much of a peak at 280nm on Nanodrop - more of a shoulder of the 260nm peak. - Dr. B 100113
TEV was expressed, and spun down. TEV will be used to purify RpFabG since FPLC combine with the Nickel column is not working.
Samples saved from summer (samples 2 a&b) were sonicated, spun down, and purified. The results of the new experimental purification method used instead of FPLC are indicated in the nanodrop measurements. Another round of RpFabG expression was also started, grown up over night, spun down and stored for later usage. The pellet weights were 1.13 g and 1.67 g respectively.
Figure 53: RpFab G Elution 3 after purifying sample a second time via Nickel column
Figure 52: RpFab G Elution 2 after purifying sample a second time via Nickel column
Figure 51: RpFab G Elution 1 after purifying sample a second time via Nickel column
Protein characterization was completed on 9/10/13. The protein sample was concentrated down to 1 mL and run through the FPLC machine. Another round of protein purification via FPLC was completed on 9/10/13. The FPLC machine on both these two rounds and the previous round skipped collection tubes and only half filled others. The graphs also indicated that there was no protein in the sample. RpFabG is 700bp ~ 300AA long, so it should have shown up towards the end of the graph. Since this method has failed three times, another purification method will be used. The protein will be purified using a nickel column multiple times and the use of protein TEV will also be involved.
Figure 50: FPLC Results RpFabG 9/10/13
Figure 49: FPLC Results RpFabG 9/9/13
Week 1 & 2 (of Aug. 28- Sept 6)
Priya - good work. Hopefully the next FPLC will be good. Dr. B 090913
FPLC
Figure 48: FPLC results of .8mL sample of protein Rp Fab G
The graph indicates that the custom buffer did not enter the purifying column until the brown line peaked. The brown line indicates conductivity which would increase when a significant concentration of salt has entered the system. The custom buffer has a NaCL concentration of 500mM. The protein Rp Fab G should be at a peak in the solid blue line, since there is no peak, but instead a drop where the custom buffer entered the system, it is likely that the peak is hidden. It should be around the red fraction number 24- 50. The protein is 700bp wich translates to 300 AA. FPLC will be re run with the correct buffer already running through the column with the remaining .8mL of concentrated elution 1 Rp Fab G protein sample.
Spinning Down and Concentrating Samples
Figure 47: Nanodrop results of combined .8mL sample of concentrated elutions 1 and 2. Protein Rp Fab G
Figure 46: Nanodrop results of combined elutions 1 after being spun down and then concentrated to 1mL. Protein Rp Fab G
Figure 45: Nanodrop results of both elution 1 samples after being combined and spun down. Protein Rp Fab G
After 12 hours of storage, Sample 1 and 2 of Elutions 1 had precipitated, so the samples were combined, spun down, nano dropped, concentrated to 1 mL, and then nano dropped again to get concentration readings. Elutions 2 were combined and concentrated together to .5 mL and then combined with .3mL of concentrated Elution 1. The .8mL sample was run through the FPLC machine.
Protein Purification
Figure 44: Elution 2 nanodrop results, RpFabG
Figure 43: Elution 1 nanodrop results, RpFabG
Protein expression, purification, and FPLC were complete this week. Elution 1 of the protein had precipitated after 12 hours of storage in the 4C fridge.
Fall 2013
Week 8
Protein Characterization
Protein characterization was started from OEV's positive clones. Pellets were spun down, re-suspended, and stored for long term use in the -80 C freezer.
Mini Prep
Figure 42: Mini prep nano drop results, Measurement 2
Figure 41: Mini prep nano drop results, Measurement 1
Figure 40: Mini-Prep Verification gel. Well 1: 1kb DNA Ladder, Well 2: Uncut plasmid control, Well 3: Cut sample. Well 4: Skip, Well 5: 1kb DNA Ladder, Well 6: Uncut plasmid control, Well 7: Cut sample.
The samples in wells 1-3 were loaded, but the positive and negative cables were connected incorrectly so that the sample ran up the gel instead of down. The cables were fixed and then wells 5-7 were loaded when the original samples were thought to have reached the wells again. Wells 5-7 served as a back up just in case the DNA had already run off the gel from lanes 1-3.
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Week 7
Cloning was attempted twice, the second attempt being somewhat successful with one colony present after one 24 hr period. The one colony was then used to continue on with the master plate protocol.
RE Digest
Figure 39: RE Digest Gel. Well 1: 100bp DNA ladder, Well 2: PMP RE Digest sample.
PCR Clean Up
Figure 38: PCR Clean up results for RE Digest sample. Measurement 2
Figure 37: PCR Clean Up for RE Digest results. Measurement 1
RE Digest
Figure 36: RE Digest, Well 1: 1kb DNA ladder, Well 2: OEV RE digest, Well 3: PMP RE Digest, Well 4: BH RE Digest. All 3 share the same target.
Week 6
Priya - include your PCR results of squared - DR. B 071713
7/13/13
Cloning plates were checked. No colonies were apparent.
7/12/13
PCR Clean Up
Since the previous cut plasmid had a concentration that was too low, 4 new samples (50uL each) were made and the combined into one 200uL sample in PCR clean up to increase the concentration.
Figure 35: PCR Clean Up results from 200uL of cut plasmid PNiC-BSa4. Measurement 2
Figure 34: PCR Clean Up results from 200uL of cut plasmid PNiC-BSa4
Two samples (50uL each) were prepared for PNiC-BSa4 in case one failed or had a concentration too low to be used. The concentrations of both samples would ideally be around 50ng/uL. Both samples indicate high purity through the 260/280 and 260/230 values. The RE Digest Gel shows that the cutting of plasmid PNiC-BSa4 was successful. With the current concentrations of the samples, cloning might be successful and will be carried out.
Figure 33: RE Digest for PNiC-BSa4 prep samples. Well 1: skip, Well 2: 1kb ladder, Well 3: Sample 1, Well 4: 1kb ladder, Well 5: Sample 2
Figure 32: Sample 2 from PniC-BSa4 Prep measurement 2
Figure 31: Sample 2 from PniC-BSa4 Prep measurement 1
Figure 30: Sample 1 from PniC-BSa4 Prep measurement 2
Figure 29: Sample 1 from PniC-BSa4 Prep measurement 1
7/10/13
PCR Clean Up Re-Do(s)
After completing PCR Clean Up correctly the concentration of the sample was over 100 ng/uL. This supports the conclusion as to why the precious sample had low concentrations.
Figure 28: Measurement 2 after PCR Clean Up (third PCR Clean Up) 7/10/13
Figure 27: Measurement 1 after PCR Clean Up (third PCR Clean Up) 7/10/13
In this PCR clean up, step 1 was skipped. In step 1, the column filter is prepared for maximum DNA binding to the membrane. Since this step was overlooked, the concentration of the sample suffered. PCR^2 was redone as well as PCR Clean Up.
Figure 26: Measurement 2 after PCR Clean Up (second PCR Clean Up) 7/10/13
Figure 25: Measurement 1 after PCR Clean Up (second PCR Clean Up) 7/10/13
PCR Overlap Re-Do
Figure 24: PCR Overlap RpFabG, Well 1: 1kb DNA ladder, Well 2: Primary PCR with original oligo Mix, Well 3: Secondary PCR with original Oligo mix, Well 4: skip, Well 5: 1kb DNA ladder, Well 6: Primary PCR with newly made Oligo mix, Well 7: Secondary PCR with newly made Oligo mix.
7/9/13
FPLC
Graph 1: FPLC Result for PfDXR sample. 7/9/13. PfDXR is at the peak around 27(red numbers).
PCR Clean Up
The concentrations of these samples could be low due to PCR^2 not being successful or because the primary and secondary PCR samples were too weak for PCR^2 to have a significant impact on the amplification of DNA present in the sample.
Figure 23: Measurement 2 after PCR Clean Up 7/9/13
Figure 22: Measurement 1 after PCR Clean Up 7/9/13
7/8/13
FPLC
Elution samples for PfDXR were spun down and concentrated into a 1mL sample.
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Week 5
Midi Prep
Figure 21: Midi-Prep PNiC BSa4 Measurement 2.
Figure 21: Midiprep PNiC BSa4 Measurement 1.
Overlap PCR
Figure 20: PCR Primer Overlap, Well 1: 1kb DNA LAdder, Well 2: Primary PCR RpFabG, Well 2: Secondary PCR RpFabG. Both are very feint. Well 4: skip, Wells 5-7 are VLG
Figure 19. PCR Primer Overlap. Well 1: 1kb DNA ladder, Well 2: Primary PCR RpFabG, Well 2: Secondar PCR RpFab G. Well 4: Skip, Wells 5-7 are VLG
Pnic-PCR
Figure 18: PNiC BSa4 PCR Results. Well 1: 100bp Ladder, Well 2: Dilution 3, Well 3: Dilution 2, Well 4: Dilution 1, Well 5: Control. Well 6-10 are VLG
Tail Primer Design
Forward Primer: 5’ TACTTCCAATCCATGATTGACCTCACGGGCAA 3’ 32 bp Content 46.9% 0 mM Mg2+ Tm 64.4oC 1.5 mM Mg2+ Tm 71.5 oC 2 mM Mg2+ Tm 72.0 oC 4 mM Mg2+ Tm 72.9 oC 6 mM Mg2+ Tm 73.4 oC
Reverse Primer: 5’ GTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’ Reverse complement it: 5’ TATCCACCTTTACTGTTAAACCATCAGCATACCAC 3’ 35 bp GC Content 40.0% 0 mM Mg2+ Tm 61.2 oC 1.5 mM Mg2+ Tm 68.8 oC 2 mM Mg2+ Tm 69.3 oC 4 mM Mg2+ Tm 70.3 oC 6 mM Mg2+ Tm 70.8 oC
Figure 17: Fab G 3-ketoacyl-(acyl-carrier-protein) reductase; GOI
FIgure 16: Acceptance Vector PNicBSa4 withy BsaI cutting locations to insert Fab G 3-ketoacyl-(acyl-carrier-protein) reductase gene. Displayed linearly.
FIgure 15: Acceptance Vector PNicBSa4 withy BsaI cutting locations to insert Fab G 3-ketoacyl-(acyl-carrier-protein) reductase gene. Displayed circularly.
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Week 4
Figure 14: MEasurement of PfDXR; max abs at 280 nm; Elution 2
Figure 16: PfDXR gel. Well 1:Empty, Well 2:100bp DNA Ladder; Well 3: Sample 0, Well 4: Sample 1; Well 5: Sample 2; Well 6: Sample 3; Well 7: Sample 4; Well 8: Sample 5; Well 9: Sample 6; Well 10: Empty
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Week 3
Figure 10: Spectrophotometer readings for PfDXR Protein expression readings
Figure 11: PMCherry PCR. Well 1 1kb DNA ladder, Well 2 Sample 1, Well 3 Sample 2, Well 4 Sample 3, Well 5 Sample 4, Well 6 Sample 5, Well 7 Sample 6, Well 8 Sample 7, Well 9 Sample 8, Well 10 Empty
Figure 12: PMCherry PCR Lane 1: 100bp DNA ladder Lanes 2-5 are Samples 1-4 respectively. Lanes 6-10 are Vicky G. lanes.
Figure 13: PCR PNiC-BSa4. LAne 1 100 bp DNA ladder, Lanes 2-5 are Samples 1-4 respectively. Lanes 6-10 are Vicky G.'s lanes.
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Week 2
Figure 6: PnIC BSa4 Elution Measurement 1
FIgure 7: PnIC BSa4 Elution Measurement 2
Figure 8: pGBR22 PCR Agrose gel. Priya wells 6-10. DNA 100bp ladder well 6, Sample A well 7, Sample B well 8, Sample C well 9, Sample D well 10
Figure 9: PCR2 PMCherry. Well 1 1kb DNA ladder, Well 2 Sample 1, Well 3 Sample 2, Well 4 Sample 3, Well 5 Sample 4, Well 6 Sample 5, Well 7 Sample 6, Well 8 Sample 7, Well 9 Sample 8, Well 10 Empty
Midi Prep DNA Sequencing Results:
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Week 1
DNA sequence results: NNNNN
Image1: DNA Sequencing Results Order 1
The DNA Sequencing results indicated that there were nucleic acids present in the sample but the specific bases were not able to be determined.
pNiC Plasmid.
Figure 1: Plate A, LB+ Kanamycin, plasmid PNiC-BSa4, 1ng
Week 15 & 16 (Dec 2 - Dec 8)
Cation Enchange
Cation Exchange was completed. Nanodrop results show that it was unsuccessful. A protein gel will be run to verify these results.Elution 4: 0% Buffer A: 100% Buffer B (1M NaCL, 20mM HEPES)
Elution 3: 25% Buffer A: 75% Buffer B
Elution 2: 50% Buffer A: 50% Buffer B
Elution 1: 75% Buffer A: 25% Buffer B
The goal concentration was to get as close to .1 mg/ml as possible. The -0.00 concentration indicates that all the contaminants were positively charged, and stuck to the negatively charged resin. This is a highly unlikely occurrence so the rest of the procedure was completed as a safety precaution.
Week 13 & 14 (Nov 18 - Dec 1)
Enzyme Assay
The enzyme Assay was unsuccessful. This could be due to contaminants in the sample. Cation exchange will be used to further purify the sample since Nickel column purification was not effective.
Protein ExpressionSmall cultures went in at 10:30AM, after 8 hours (6:30PM) no growth was apparent. The cultures were left in the shaking incubator over night to see if they needed more time to grow, or if the bacterial colonies were dead. At 7:07, small culture tubes were made for the second plate of bacterial colonies with the RpFabG insert to determine if the plate was still viable. All samples grew up overnight, expression protocol was continued. Two 500mL flasks of LB were used. Flask A failed to grow while Flask B was successful. Sample B was sonicated then sun down, sterile filtered, and purified with the Nickel column. A protein gel was run to determine the purity. Elution 1 had a yield of 17.31mg in 2mL of buffer and Elution 2 had a yield of 11.2mg in 4mL of buffer. Elutions were concentrated into 1 mL and then .5mL was stored with glycerol and .5mL was snap frozen.
Week 11 & 12 (Nov 4 - Nov 17)
Great work Priya, nice assays. -UM
Enzyme Assays
Conditions in each of the enzyme assays (1-6) had different conditions. Enzyme assay 1 (Green) began at the same conditions as the assays conducted in figure 64, but was unsuccessful. Since the assay was unsuccessful, calculations were adjusted to account for more enzyme. In assay 2 (Blue) 30 uL of enzyme was used at the start, enzyme was added multiple times in 5 uL increments. Enzyme assay 3 (purple) was adjusted to being with 50 uL of enzyme. Assay 4 (orange) Started with 70 uL of enzyme and more was added multiple times in 10 uL increments. Assay 5 (pink) started with 100uL of enzyme and additional enzyme (50 uL) was added once. Enzyme assay 6 (red) had enzyme added in 50 uL increments for a total of 500 uL. Assay 1-4 were with sample stored in the -80C freezer while assay 5r and 6 were using sample stored with glycerol. All enzyme assays were unsuccessful. This could be attributed to having a contaminated sample or samples with low concentrations. There is a possibility that the enzyme can not be stored for long periods of time and still be functional.
Enzyme Assays
Enzyme assay 1 (Orange) began at the same conditions as the assays conducted in figure 64, but enzyme was added multiple times in 5uL increments. In assay 2 (Pink) 20 uL of enzyme was used at the start. Enzyme assay 3 (green) had the same starting conditions as assay 2 but enzyme (5uL) was added one additional time. Assay 4 (blue) Tris buffer was used instead of HEPES since the assays were not successful. This assay showed that NADPH was being oxidized by the enzyme (RpFabG) since absorbance was decreasing.
Virtual Screening
The top 5 ligands from the CB_306 library with a logP below 3 were ordered.
Week 9 & 10 (Oct 21 - 3- Nov)
Great work! It would be good to include the results table from your ligand virtual screening run, too! -Suman 11/5/13
Virtual Screening
Enzyme Assay
The orange line is the end of the third assay. The green line is NADPH, buffer and water, the blue is Buffer, water, NADPH, and enzyme. The red line is enzyme,ACC, NADPH, water, and buffer. These results show that NADPH is the smaller hill at about 340nm. The next steps will be to attempt to replicate this data and then to vary the amount of NADPH while keeping the amount of AAC constant. Then vary the amount of AAC while keeping the amount of NADPH constant in several enzyme assays.
The absorbance of NADPH decreased as the reaction progressed. NADPH was oxidized to NADP. The graph indicated the assay worked.
Dilutions for NADPH and AAC were calculated and prepared. Enzyme assay calculations were completed and three assays were conducted.
Virtual Screening
Five negative controls and 10 positive controls were obtained using PDB. The SDF files were concatenated into a control library. The library was then lig-prepped using ICM since Maestro was still down and docked using GOLD. The control ligand docking was analyzed and confirmed as accurate. Script files were made to run screening jobs on the Hit Finder library, NIH Clinical, Cb 306, and In House libraries.
The pdb structure of RpFabG was obtained (3F9I). No ligands were docked, so NADPH was extracted from FabG from another organism. An attempt to ligand prep NADPH was made but ICM and Maestro were down. After numerous attempts at different times on different days, NADPH was prepared using ICM since Maestro was still down. NADPH was then docked multiple times using ICM with each attempt resulting in various errors or a low docking score. GOLD was then used to dock NADPH into the active site, generating 30 poses.
Week 7 & 8 (Oct 7 - Oct 20)
Good captions and brief analysis of your data. Where are you with virtual work? Thank you. -Max 10/21/13RpFabG Sonication, Spin Down, Purification, Characterization, Concentration, and Storage**
The two samples saved from the previous week were sonicated, spun down, filtered, and then purified. The concentrations of these elutions are relatively high when compared to past elution samples for RpFabG. This can be attributed to using the custom made HEPES buffer with a high concentration of imidazole instead of the elution buffer. A characterization protein gel was run containing elution 1 and 2 samples. The purified samples were stored for 2 days.The samples were then concentrated to about 2mL, split into two 1mL samples that were stored by snap freezing with liquid nitrogen and glycerol respectively. There is still contamination in sample, but FPLC was skipped this time since all previous attempts failed.
Week 5 & 6 (Sept 23 - Oct 6)
Good Captions but try to add an analysis of your data. Thank you. -Max 10/07/201310/1/13
Virtual Screening
Positive and Negative controls were found for target. Since NADPH was not docked in the original crystal structure, similar structures of FabG were used to dock NADPH and define the active site after completing the ligand prep protocol.
RpFabG Expression
RpFabG was expressed using the new expression protocol, Option A. The sample was stored to be sonicated.
9/27-28/13
Protein Expression**
RpFabG sample that was stored after sonication was spun down, purified using the nickel column, and then purified through FPLC. FPLC result show a minimal amount of protein, but not enough to be purified. RpFabG will be expressed again and samples will be purified in the room temperature FPLC machine using a nickel column.
Week 3 & 4 (Sept 9 - Sept 22)
9/16/13Priya - Good work on purifications. what does your gel look like for these? There doesn't seem to be much of a peak at 280nm on Nanodrop - more of a shoulder of the 260nm peak. - Dr. B 100113
TEV was expressed, and spun down. TEV will be used to purify RpFabG since FPLC combine with the Nickel column is not working.
Samples saved from summer (samples 2 a&b) were sonicated, spun down, and purified. The results of the new experimental purification method used instead of FPLC are indicated in the nanodrop measurements. Another round of RpFabG expression was also started, grown up over night, spun down and stored for later usage. The pellet weights were 1.13 g and 1.67 g respectively.
Protein characterization was completed on 9/10/13. The protein sample was concentrated down to 1 mL and run through the FPLC machine. Another round of protein purification via FPLC was completed on 9/10/13. The FPLC machine on both these two rounds and the previous round skipped collection tubes and only half filled others. The graphs also indicated that there was no protein in the sample. RpFabG is 700bp ~ 300AA long, so it should have shown up towards the end of the graph. Since this method has failed three times, another purification method will be used. The protein will be purified using a nickel column multiple times and the use of protein TEV will also be involved.
Week 1 & 2 (of Aug. 28- Sept 6)
Priya - good work. Hopefully the next FPLC will be good. Dr. B 090913FPLC
The graph indicates that the custom buffer did not enter the purifying column until the brown line peaked. The brown line indicates conductivity which would increase when a significant concentration of salt has entered the system. The custom buffer has a NaCL concentration of 500mM. The protein Rp Fab G should be at a peak in the solid blue line, since there is no peak, but instead a drop where the custom buffer entered the system, it is likely that the peak is hidden. It should be around the red fraction number 24- 50. The protein is 700bp wich translates to 300 AA. FPLC will be re run with the correct buffer already running through the column with the remaining .8mL of concentrated elution 1 Rp Fab G protein sample.
Spinning Down and Concentrating Samples
After 12 hours of storage, Sample 1 and 2 of Elutions 1 had precipitated, so the samples were combined, spun down, nano dropped, concentrated to 1 mL, and then nano dropped again to get concentration readings. Elutions 2 were combined and concentrated together to .5 mL and then combined with .3mL of concentrated Elution 1. The .8mL sample was run through the FPLC machine.
Protein Purification
Fall 2013
Week 8
Protein Characterization
Protein characterization was started from OEV's positive clones. Pellets were spun down, re-suspended, and stored for long term use in the -80 C freezer.Mini Prep
The samples in wells 1-3 were loaded, but the positive and negative cables were connected incorrectly so that the sample ran up the gel instead of down. The cables were fixed and then wells 5-7 were loaded when the original samples were thought to have reached the wells again. Wells 5-7 served as a back up just in case the DNA had already run off the gel from lanes 1-3.
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Week 7
Cloning was attempted twice, the second attempt being somewhat successful with one colony present after one 24 hr period. The one colony was then used to continue on with the master plate protocol.RE Digest
PCR Clean Up
RE Digest
Week 6
Priya - include your PCR results of squared - DR. B 0717137/13/13
Cloning plates were checked. No colonies were apparent.
7/12/13
PCR Clean Up
Since the previous cut plasmid had a concentration that was too low, 4 new samples (50uL each) were made and the combined into one 200uL sample in PCR clean up to increase the concentration.DNA Sequencing Results for PNiC-BSa4
Filtered DNA Sequence:NNNNNNNNGNNNACTTTAGNNGAGATATACATATGCACCATCATCATCATCATTCTTCTG GTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGAGACCGACGTCCACATAT ACCTGCCGTTCACTATTATTTAGTGAAATGAGATATTATGATATTTTCTGAATTGTGATT AAAAAGGCAACTTTATGCCCATGCAACAGAAACTATAAAAAATACAGAGAATGAAAAGAA ACAGATAGATTTTTTAGTTCTTTAGGCCCGTAGTCTGCAAATCCTTTTATGATTTTCTAT CAAACAAAAGAGGAAAATAGACCAGTTGCAATCCAAACGAGAGTCTAATAGAATGAGGTC GAAAAGTAAATCGCGCGGGTTTGTTACTGATAAAGCAGGCAAGACCTAAAATGTGTAAAG GGCAAAGTGTATACTTTGGCGTCACCCCTTACATATTTTAGGTCTTTTTTTATTGTGCGT AACTAACTTGCCATCTTCAAACAGGAGGGCTGGAAGAAGCAGACCGCTAACACAGTACAT AAAAAAGGAGACATGAACGATGAACATCAAAAAGTTTGCAAAACAAGCAACAGTATTAAC CTTTACTACCGCACTGCTGGCAGGAGGCGCAACTCAAGCGTTTGCGAAAGAAACGAACCA AAAGCCATATAAGGAAACATACGGCATTTCCCATATTACACGCCATGATATGCTGCAAAT CCCTGAACAGCAAAAAAATGAAAAATATAAAGTTCCTGAGTTCGATTCGTCCACAATTAA AAATATCTCTTCTGCAAAAGGCCTGGACGTTTGGGACAGCTGGCCATTACAAAACACTGA CGGCACTGTCGCAAACTATCACGGCTACCACATCGTCTTTGCATTAGCCGGAGATCCTAA AAATGCGGATGACACATCGATTTACATGTTCTATCAAAAAGTCGGCGAAACTTCTATTGA CAGCTGGAAAACGCTGGNCNNCGTCTTTNAAGACAGCGACAANTCGATGCNNTGATNCTA TCCTAAAANACCAAACNNANAANGGNCAGGNTCANCNNCATTTACATCTGANNNAANNNN NTNNTTCTNNNNTGATTTNNNCNGNNNNNNNNGNNANNNANNNCTNNNNNNNNNNNNNNN CNNATCNNNATCNNANNNNNNTTNNANNNNNNNNNNNNNNNNNNNNNNNANNNNNNNNNN
NNNNANNNNNNNANNNNNNGNANNNNNNNNNNNAANNNNNNNNNNNNNNNNNNNN
Nucleotide Blast Results for PNiC-BSa4
7/11/13
PNiC-BSa4 Cloning
Two samples (50uL each) were prepared for PNiC-BSa4 in case one failed or had a concentration too low to be used. The concentrations of both samples would ideally be around 50ng/uL. Both samples indicate high purity through the 260/280 and 260/230 values. The RE Digest Gel shows that the cutting of plasmid PNiC-BSa4 was successful. With the current concentrations of the samples, cloning might be successful and will be carried out.7/10/13
PCR Clean Up Re-Do(s)
After completing PCR Clean Up correctly the concentration of the sample was over 100 ng/uL. This supports the conclusion as to why the precious sample had low concentrations.In this PCR clean up, step 1 was skipped. In step 1, the column filter is prepared for maximum DNA binding to the membrane. Since this step was overlooked, the concentration of the sample suffered. PCR^2 was redone as well as PCR Clean Up.
PCR Overlap Re-Do
7/9/13
FPLC
PCR Clean Up
The concentrations of these samples could be low due to PCR^2 not being successful or because the primary and secondary PCR samples were too weak for PCR^2 to have a significant impact on the amplification of DNA present in the sample.7/8/13
FPLCElution samples for PfDXR were spun down and concentrated into a 1mL sample.
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Week 5
Midi Prep
Overlap PCR
Pnic-PCR
Tail Primer Design
Forward Primer:5’ TACTTCCAATCCATGATTGACCTCACGGGCAA 3’ 32 bp
Content 46.9%
0 mM Mg2+ Tm 64.4oC 1.5 mM Mg2+ Tm 71.5 oC 2 mM Mg2+ Tm 72.0 oC
4 mM Mg2+ Tm 72.9 oC 6 mM Mg2+ Tm 73.4 oC
Reverse Primer:
5’ GTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’
Reverse complement it:
5’ TATCCACCTTTACTGTTAAACCATCAGCATACCAC 3’ 35 bp
GC Content 40.0%
0 mM Mg2+ Tm 61.2 oC 1.5 mM Mg2+ Tm 68.8 oC 2 mM Mg2+ Tm 69.3 oC
4 mM Mg2+ Tm 70.3 oC 6 mM Mg2+ Tm 70.8 oC
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Week 4
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Week 3
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Week 2
Midi Prep DNA Sequencing Results:
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Week 1
DNA sequence results: NNNNN
The DNA Sequencing results indicated that there were nucleic acids present in the sample but the specific bases were not able to be determined.
pNiC Plasmid.